The cell cycle for replicating cells can be divided into two periods: (1) the cell division period, when the cell divides and separates, with each daughter cell receiving identical copies of the DNA; and (2) the period of growth, known as the interphase period. For the cell cycle of eucaryotes, the cell division period is termed the M (mitotic) period. The interphase period in eucaryotes is further divided into three successive phases: G1 (gap 1) phase, which directly follows the M period; S (synthetic) phase, which follows G1; and G2 (gap 2) phase, which follows the S phase, and immediately precedes the M period. During the two gap phases no net change in DNA occurs, though damaged DNA may be repaired. On the other hand, throughout the interphase period there is continued cellular growth and continued synthesis of other cellular components. Towards the end of the G1 phase, the cell passes a restrictive point and becomes committed to duplicate its DNA. At this point, the cell is also committed to divide. During the S phase, the cell replicates DNA. The net result is that during the G2 phase, the cell contains two copies of all of the DNA present in the G1 phase. During the subsequent M period, the cells divide with each daughter cell receiving identical copies of the DNA. Each daughter cell starts the next round of the growth cycle by entering the G1 phase.
The G1 phase represents the interval in which cells respond maximally to extracellular signals, including mitogens, anti-proliferative factors, matrix adhesive substances, and intercellular contacts. Passage through the restrictive point late in G1 phase defines the time at which cells lose their dependency on mitogenic growth factors for their subsequent passage through the cycle and, conversely, become insensitive to anti-proliferative signals induced by compounds such as transforming growth factor, cyclic AMP analogs, and rapamycin. Once past the restrictive point, cells become committed to duplicating their DNA and undergoing mitosis, as noted above, and the programs governing these processes are largely cell autonomous. (See generally, Darnell et al. (1986) in Molecular Cell Biology, pp. 146-148, Scientific American Books, New York.)
Regulation of the human cell cycle requires the periodic formation, activation, and inactivation of protein kinase complexes that consist of a regulatory (cyclin) subunit and a catalytic (cyclin dependent kinase or cdk) subunit. Cell cycle-dependent fluctuations in the levels of many of the cyclin proteins contribute to the activation of these protein kinase complexes. For example, cyclin B participates in the regulation of the G2/M transition by its association with its catalytic subunit, p34.sup.cdc2, whereas cyclin A, in complexes with both p34.sup.cdc2 and cdk2, is essential for the completion of S-phase and entry into G2-phase. Complexes formed between the D-type cyclins and either cdk4 or cdk6 integrate growth factor signals and the cell cycle, allow cells to progress through G1-phase. This particular cell cycle pathway is specifically altered during tumorigenesis, presumably due to its role in responses to mitogenic stimulation. Alterations have been identified in many components of this pathway, including the D-type cyclins, cyclin dependent protein kinases, and cyclin dependent kinase inhibitors (CKIs). Another G1-phase cyclin, cyclin E, in conjunction with its catalytic subunit cdk2, appears to be essential for progression from G1-phase into S-phase and the initiation of DNA replication. Cyclin E and cdk2 do not appear to be directly targeted during tumorigenesis, quite possibly due to their essential nature. (See generally, Sherr, Cell 79:551-555 (1994)) and Sherr, Cell 73:1059-1065 (1993)).
A class of novel polypeptides that are collectively known as cdk inhibitors (CKIs) can negatively regulate cyclin/cdk activity by associating with these complexes. These so-called "cell cycle brakes" act to inhibit cyclin/cdk complexes by binding specifically to either the cyclin or the cdk, but generally not both. CKI activity is cell cycle regulated allowing these proteins to function as inhibitors of their cognate cyclin/cdk complexes for very limited periods during the cell cycle. The cdk inhibitors isolated thus far include p21.sup.Cip1,Waf1,Sdi1,Cap20, p27.sup.Kip1, p57.sup.Kip2, and a small family of inhibitors of cdk4 (INK4), which include p16.sup.INK4a, p15.sup.INK4b, p18.sup.INK 4c, and p19.sup.INK4d. In some cases (i.e., p.sub.21.sup.Cip1,Waf1,Sdi1,Cap20) this inhibitory activity may be conditional, and these proteins may also act to positively regulate cyclin/cdk complexes by functioning as "bridge" molecules that maintain complex formation and enzyme activity. The p21, p27, and p57 proteins have been found in association with multiple cyclins (including D, E, and A), while the INK4 inhibitors specifically interact with complexes containing the D-type cyclins and cdk4 or cdk6. Because these proteins are important in cell cycle control, many are targeted for inactivation by both tumor viruses and genetic alterations during oncogenesis. Loss of specific CKI function can result in unregulated DNA replication, and aid in the generation of further mutations due to accumulating DNA damage. To date, mutations and/or deletions of the p16.sup.INK4a gene have been observed in a broad range of tumor types, strongly supporting its role as a tumor suppressor. (See generally, Sherr et al., Genes & Devel. 9:1149-1163 (1995)) and Morgan, Nature 374:131-134 (1995).) Cyclin C was originally isolated from both human and Drosophila cDNA libraries by virtue of its ability to complement a CLN1-3 defective S. cerevisiae strain (Lew et al., Cell 66:1197-1206 (1991); Leopold et al., Cell 66:1207-1216 (1991); Lahue et al., (1991)). The CLN1-3 yeast cyclins function during the G1-phase of the cell cycle by helping convey external growth signals to the nucleus and promote cell cycle progression (Cross, Mol. Cell Biol. 10:6482-6490 (1990). Thus, it was postulated that cyclin C was, itself, a G1-phase cyclin regulatory partner. The recent discovery of a cdk partner for cyclin C, cdk8, indicates that, like other G1-phase cyclins, cyclin C functions to regulate a specific cdk, in a cell cycle dependent manner (Tassan et al., Proc. Natl. Acad. Sci., USA 92: 8871-8875 (1995)). Cyclin C not only associates with cdk8 in vitro, thereby activating the kinase, but also associates with cdk8 in vivo. Furthermore, the high degree of sequence identity between mammalian cyclin C and cdk8 with the S. cerevisiae SRB10 and SRB11 gene products, indicate that this complex plays an important role in the regulation of transcription (O'Neill et al., Nature, 374:121-122 (1995); Liao et al., Nature 374:193-196 (1995)). In yeast, the SRB10 and SRB11 gene products, a cdk and a regulatory cyclin partner respectively, are associated in vivo with RNA polymerase II. This cyclin/cdk complex is involved in both positive and negative regulation of transcription. The complex also may play a particularly important role in relaying extracellular growth signals to the transcription apparatus by either directly, or indirectly, phosphorylating the C-terminal domain (CTD) of RNA polymerase II (Tassan et al., supra).
The human cdk 8 catalytic partner of cyclin C maps to human chromosome 13q12, a region associated with the BRCA2 breast cancer susceptibility gene, but the significance of this observation is unknown. The CCNC gene encoding human cyclin C has recently been cloned and localized to human chromosome 6q21 (Demetrick et al., Cytogenet. Cell Genet. 69:190-192 (1995); Li et al. (1996a)). This region of chromosome six is often deleted or altered during tumorigenesis, suggesting that the CCNC gene might be a candidate tumor suppressor (Kowalczyk et al., (1985); Prigogina et al. (1988)). The integrity of the CCNC gene was examined in a subset of acute lymphoblastic leukemias (ALLs) with 6q21 deletions, and one allele of this gene was consistently deleted (&gt;90% of primary tumors) (Li et al., (1996a)). However, careful examination of the remaining CCNC allele by single-strand conformational polymorphism (SSCP) revealed no further physical alterations. Similar results involving the frequent deletion of cyclin dependent protein kinase inhibitors, such as p.sub.27.sup.Kip1, have been demonstrated by others. It is possible that these genes exert their effects in tumors due to haploinsufficiency of the corresponding proteins, or that they are not the actual tumor suppressors, but share close physical linkage with the target genes (Pietenpol et al., Cancer Res. 55:1206-1210 (1995); Li et al. (1996)).
The existence of a specific and unique cyclin dependent protein kinase partner, cdk8, for cyclin C indicates that cyclin C functions like other cyclins, i.e., to regulate a kinase catalytic domain. It has been proposed that the cyclin C-cdk8 complex regulates RNA transcription during the cell cycle. However, to date, no factor analogous to a cdk inhibitor has been identified that, in turn, specifically regulates cyclin C/cdk8 complex activity.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.